BIOLOGY OF REPRODUCTION 84, 255–265 (2011)
Published online before print 22 September 2010.
DOI 10.1095/biolreprod.110.086942
Altered Spatiotemporal Expression of Collagen Types I, III, IV, and VI
in Lpar3-Deficient Peri-Implantation Mouse Uterus1
Honglu Diao,3 John D. Aplin,5 Shuo Xiao,3,4 Jerold Chun,6 Zuguo Li,3 Shiyou Chen,3 and Xiaoqin Ye2,3,4
Department of Physiology and Pharmacology,3 College of Veterinary Medicine, and Interdisciplinary Toxicology
Program,4 University of Georgia, Athens, Georgia
Maternal and Fetal Health Research Group,5 Manchester Academic Health Sciences Centre, St. Mary’s Hospital,
University of Manchester, Manchester, United Kingdom
Department of Molecular Biology,6 Dorris Neuroscience Center, The Scripps Research Institute, La Jolla, California
Lpar3 is upregulated in the preimplantation uterus, and
deletion of Lpar3 leads to delayed uterine receptivity in mice.
Microarray analysis revealed that there was higher expression of
Col3a1 and Col6a3 in the Preimplantation Day 3.5 Lpar3/
uterus compared to Day 3.5 wild-type (WT) uterus. Since
extracellular matrix (ECM) remodeling is indispensable during
embryo implantation, and dynamic spatiotemporal alteration of
specific collagen types is part of this process, this study aimed to
characterize the expression of four main uterine collagen types:
fibril-forming collagen (COL) I and COL III, basement membrane COL IV, and microfibrillar COL VI in the peri-implantation WT and Lpar3/ uterus. An observed delay of COL III and
COL VI clearance in the Lpar3/ uterus may be associated with
higher preimplantation expression of Col3a1 and Col6a3. There
was also delayed clearance of COL I and delayed deposition of
COL IV in the decidual zone in the Lpar3/ uterus. These
changes were different from the effects of 17beta-estradiol and
progesterone on uterine collagen expression in ovariectomized
WT uterus, indicating that the altered collagen expression in
Lpar3/ uterus is unlikely to be a result of alterations in ovarian
hormones. Decreased expression of several genes encoding
matrix-degrading metallo- and serine proteinases was observed
in the Lpar3/ uterus. These results demonstrate that pathways
downstream of LPA3 are involved in the dynamic remodeling of
ECM in the peri-implantation uterus.
collagen types I, III, IV, and VI, decidua, endometrium, female
reproductive tract, implantation, lysophosphatidic acid receptor
3, pregnancy, uterus
INTRODUCTION
Collagens, the main protein components of extracellular
matrix (ECM), provide tissues with their strength and shape. In
addition to their structural functions, collagens act as ligands
for cell membrane receptors (e.g., integrins and annexin A5)
through which they mediate cellular functions, such as growth,
survival, differentiation, adhesion, and migration [1, 2]. There
1
Supported by startup funding from University of Georgia to X.Y. and by
National Institutes of Health grant HD050685 to J.C.
2
Correspondence: Xiaoqin Ye, Department of Physiology and Pharmacology, College of Veterinary Medicine & Interdisciplinary Toxicology
Program, University of Georgia, Athens, GA 30602.
FAX: 706 542 3015; e-mail: ye@uga.edu
Received: 28 June 2010.
First decision: 2 August 2010.
Accepted: 16 September 2010.
Ó 2011 by the Society for the Study of Reproduction, Inc.
eISSN: 1529-7268 http://www.biolreprod.org
ISSN: 0006-3363
255
Downloaded from www.biolreprod.org.
are at least 28 different collagen subtypes classified into eight
subfamilies: fibril-forming collagen (e.g., collagen [COL] I);
basement membrane collagen (e.g., COL IV); beaded filamentforming collagen (e.g., COL VI); anchoring fibril-forming
collagen (e.g., COL VII); fibril-associated collagens with
interruptions in triple helix (COL IX); hexagonal networkforming collagens (e.g., COL X); transmembrane collagens
(e.g., COL XIII); and multiplexins (e.g., COL XVIII) [2].
Uterine collagen content alters during pregnancy and
postpartum involution [3]. In the embryo implantation phase
of early pregnancy, progressive differentiation of fibroblastlike stromal cells into hypertrophic polygonal decidual cells
correlates with profound alterations in collagen gene expression [4–7]. It has been demonstrated that different collagen
types have distinct spatiotemporal expression patterns during
implantation. The major fibril-forming COL I, III, and V are all
reduced in implantation sites compared to non-implantation
sites in the rat uterus [4], mainly because they progressively
disappear from the primary and secondary decidual zones from
Day 5.5 to Day 7.5 (Day 0 as mating night) [5–7]. Their
expression patterns in the outer nondecidualized stroma and
myometrial tissues do not seem to change during this period [5,
6]. COL IV is also a main endometrial ECM component [8],
disappearing in the luminal epithelial basement membrane at
implantation site [9], but accumulating in the decidual zone
[10]. COL VI is lost from rat stromal cells undergoing
decidualization [11]. It has been suggested that abnormally
increased deposition of collagen might impair uterine function,
possibly by interfering with vascularization [12] or retarding
remodeling events at implantation.
Uterine ECM remodeling is regulated by hormones and
local factors; 17b-estradiol (E2) can inhibit the loss of collagen
from the involuting rat uterus in vivo [13], which reflects the
inhibitory role of E2 on collagenase activity [14]. Progesterone
(P4) was also shown to have an inhibitory effect on collagenase
activity, preventing collagen degradation in postpartum rat
uterine explants [15]. RU486, an antagonist and partial agonist
of progesterone receptor (PR), can reduce COL IV expression
in the Rhesus monkey decidua and villus during the first
trimester of pregnancy [16]. It has been suggested that the
spatiotemporal variation of PR and estrogen receptor in the
uterus and cervix controls P4 and E2 action at parturition [17,
18], perhaps by regulating matrix metalloproteinases (MMPs),
their inhibitors, and matrix-degrading serine proteinases [1, 19–
23].
Lysophosphatidic acid (LPA) is a small lipid signaling
molecule. Several reports have revealed potential roles of LPA
on collagen regulation. LPA can induce collagen matrix
contraction [24], and has a potential role in promoting wound
healing [25], in which ECM deposition is a key event. LPA can
ABSTRACT
256
DIAO ET AL.
also counteract transforming growth factor-b-induced COL I
mRNA stabilization in dermal fibroblasts, therefore potentially
preventing excessive scarring [26]. On the other hand, LPA
may increase the invasiveness of ovarian cancer by upregulating MMP activity [27].
LPA functions through its G protein-coupled receptors [28,
29]. It was previously demonstrated that deletion of Lpar3 leads
to implantation defects [30, 31]. Since uterine Lpar3 expression
peaks at Preimplantation Day 3.5, this day was chosen to screen
differentially expressed genes in Lpar3/ uterus. Microarray
results indicated higher Col3a1 and Col6a3 mRNA levels in the
Preimplantation Day 3.5 Lpar3/ uterus. This led us to
investigate the expression of four representative collagen types
in the uterus: fibril-forming COL I and COL III, basement
membrane COL IV, and microfibril-forming COL VI, in the
peri-implantation wild-type (WT) and Lpar3/ uterus.
MATERIALS AND METHODS
Reagents
Animals
WT and Lpar3/ mice (129/SvJ and C57BL/6 mixed background) were
generated from a colony at the University of Georgia, which was originally
derived from our colony at the Scripps Research Institute [30]. They were
genotyped as previously described [30]. The mice were housed in
polypropylene cages with free access to regular food and water from water
sip tubes in a reverse-osmosis system. The animal facility is on a 12L:12D
(0600–1800 h) at 23 6 18C with 30%–50% relative humidity. All methods
used in this study were approved by the Animal Subjects Programs of the
University of Georgia and conformed to National Institutes of Health guidelines
and public law.
Total RNA Isolation, Microarray Analysis,
and Real-Time PCR
To isolate total RNA for microarray analysis and real-time PCR, Day 3.5
WT and Lpar3/ uterine horns were powdered in liquid nitrogen with a mortar
and pestle. The powdered sample was quickly transferred to Trizol for total
RNA isolation. Microarray analysis using mouse Illumina Chip was performed
at the Genomics and Proteomics Core Facility, Medical College of Georgia, as
previously described [32], with three replicates in each group. For real-time
PCR, cDNA was transcribed from 1 lg of total RNA using Superscript III
reverse transcriptase with random primers. Real-time PCR reactions were
performed in 384-well plates using Sybr-Green I intercalating dye on ABI 7900
(Applied Biosystems). The primers (Integrated DNA Technologies) were:
glyceraldehyde 3-phosphate dehydrogenase (Gapdh) e3F1, 5 0 -GCCGA
GAATGGGAAGCTTGTCAT-3 0 ; Gapdh e4R1, 5 0 -GTGGTTCACACCCAT
CACAAACAT-3 0 ; Col3a1 e2F1, 5 0 -GATGTCTGGAAGCCAGAAC-3 0 ; Col3a1 e4R1, 5 0 -TCACCATTTCTCCCAGGAA-3 0 ; Col6a3 e8F1, 5 0 GTCCGAACTGAGTGAGACAG-3 0 ; Col6a3 e9R1, 5 0 -CTGGTACAACTT
CAGCGTGT-3 0 . Cma1 e3F1, 5 0 -GTCCACGACATCATGCTACT-3 0 ; Cma1
e4R1, 5 0 -GCTCCTGGAGTCTCATCTTT-3 0 ; Mcp4 e2F1, 5 0 -AGAGAG
GGTTCACAGCTACC-3 0 ; Mcp4 e4R1, 5 0 -GGCCTCTTTATCCATGATTC
-3 0 ; Mcp6 e3F2, 5 0 -CAGCTTCGTGAGCAGTATCT-3 0 ; Mcp6 e4R2, 5 0 GTATTTCCAGCACACAGCAT-3 0 ; Mmp3 e4F1, 5 0 -AATGGAGATGCT
CACTTTGA-3 0 ; Mmp3 e7R1, 5 0 -AGGAGTCCTGAGAGATTTGC-3 0 ;
Mmp7 e2F1, 5 0 -TCACTAATGCCAAACAGTCC-3 0 ; Mmp7 e5R1, 5 0 TGACTCAGACCCAGAGAGTG-3 0 ; hypoxanthine phosphoribosyltransferase
1 (Hprt1) e3F1, 5 0 -GCTGACCTGCTGGATTACAT-3 0 ; and Hprt1 e4/5R1, 5 0 CAATCAAGACATTCTTTCCAGT-3 0 .
Histology
Fixed Day 4.5 WT and Lpar3/ uterine horns were embedded in paraffin
and sectioned (4 lm) longitudinally in order to increase the chance of obtaining
sections with embryo(s), especially from Lpar3/ uteri that didn’t show
implantation sites at Day 4.5 [30]. Sections were deparaffinized, rehydrated,
and stained with hematoxylin and eosin, as previously described [33].
Immunofluorescence
Young virgin (2–4 mo old) WT and Lpar3/ females were mated naturally
with WT stud males and checked for a vaginal plug the next morning. The day
on which a vaginal plug was identified was designated as Day 0.5 (mating night
as Day 0). Uterine tissues were collected between 1100 and 1200 h on Days 0.5,
3.5, 4.5, and 5.5, as well as between 2300 and 2400 h on Day 5.0. Day 0.5
females were euthanized with CO2 inhalation, and both uterine horns were
quickly removed and snap frozen on dry ice. Oviducts from these mice were
dissected for the presence of eggs to determine the pregnancy status. Parts of
uterine horns from euthanized Day 3.5 WT and Lpar3/ females were quickly
removed and frozen on dry ice for immunofluorescence, and the remainder
flushed with 13 PBS (to determine the status of pregnancy and to remove
embryos before examination of uterine gene expression) and frozen on dry ice
for microarray analysis and real-time PCR. The uterine horns from Day 4.5, 5.0,
and 5.5 WT and Lpar3/ females were flash frozen. Sections with embryo(s)
were used for immunofluorescence. Uterine horns from different Day 4.5 WT
and Lpar3/ females were fixed in 10% formalin for 24 h at room temperature
for histology. At least three pregnant mice were included in each group.
Frozen uterine horns from Days 0.5, 3.5, and 5.5 WT and Lpar3/ females
were cross-sectioned (10 lm) with the Day 5.5 sections crossing implantation
sites. Frozen uterine horns from Days 4.5 and 5.0 WT and Lpar3/ females
were sectioned (10 lm) longitudinally in order to increase the chance of
obtaining sections with embryo(s). Only the sections with embryo(s) from Day
4.5, 5.0, and 5.5 were used in the study. The frozen uterine sections on
superfrost plus slides were fixed in freshly prepared 4% paraformaldehyde in
0.02 M PBS at room temperature (RT) for 20 min. The slides were washed in
PBS three times for 5 min each. Sections for COL I and COL III (but not COL
IV or COL VI) immunofluorescence underwent antigen retrieval before they
were subsequently incubated in 0.2% Triton-X100/PBS for 20 min, and washed
three times in PBS for 5 min each. Nonspecific staining was blocked with 10%
rabbit (for COL I and COL III) or goat (for COL IV and COL VI) serum in PBS
for 60 min at 378C. Sections were incubated with primary goat anti-collagen
antibody (1:25 for COL I and COL III) or rabbit anti-collagen antibody (1:400
for COL IV, and 1:500 for COL VI), and, in a few cases, with rat anti-PECAM
(1:200) also, in blocking reagent at 48C overnight. They were washed three
times in PBS for 10 min each, incubated with Alexa Fluor 488 rabbit anti-goat
IgG, Alexa Fluor 488 goat anti-rabbit IgG, or goat anti-rat Cy3 (1:200) in 1%
BSA/PBS for 60 min at RT, then washed in PBS twice for 5 min each. They
were then incubated with DAPI in PBS for 15 min at 378C, and washed in PBS
three times for 5 min each. The sections were mounted in Vectashield. Sections
for negative control were processed exactly the same, except that the primary
antibodies were replaced with goat IgG (for COL I and COL III) or rabbit IgG
(for COL IV and COL VI).
Ovariectomy and Hormonal Treatment
Statistical Analyses
WT virgin females (6 wk old) were ovariectomized and allowed to recover
for 2 wk to eliminate ovarian steroids from the circulation. The ovariectomized
Statistical analyses were performed using two-tail, unequal variance
Student t-test. The significant level was set at P , 0.05.
Mating and Uterine Tissue Collection
Downloaded from www.biolreprod.org.
The following reagents were obtained as indicated in parentheses: TRIZOL
and Superscript III (Invitrogen, Carlsbad, CA); dNTPs (Biomega, San Diego,
CA); Taq DNA polymerase (Lucigen, Middleton, WI); E2 and progesterone
(P4) (Sigma-Addrich, St. Louis, MO); superfrost plus slides (Fisher Scientific,
Pittsburgh, PA); power SYBR green PCR master and 384-well plates (Applied
Biosystems, Carlsbad, CA); goat anti-COL I and anti-COL III antibodies
(SouthernBiotech, Birmingham, AL); rabbit anti-COL IV and anti-COL VI
antibodies (Abcam, Cambridge, MA); goat IgG and rabbit IgG (Santa Cruz
Biotechnology, Santa Cruz, CA); rat-anti-platelet/endothelial cell adhesion
molecule (PECAM) and goat anti-rat Cy3 (gifts from Dr. Lianchun Wang at
University of Georgia); Alexa Fluor 488 goat anti-rabbit IgG and Alexa Fluor
488 rabbit-anti goat IgG (Invitrogen, Carlsbad, CA); DAPI (4 0 ,6 0 -diamidino-2phenylindole) (MP Biomedicals, Solon, OH); Vectashield (Vector Labs,
Burlingame, CA).
mice received s.c. injection of vehicle (sesame oil), E2 (100 ng), or P4 (2 mg) in
0.1 ml vehicle daily. The mice were dissected 6, 24, or 72 h after the first
injection. Uterine tissues were quickly removed and frozen on dry ice for
immunofluorescence.
COLLAGEN EXPRESSION IN PERI-IMPLANTATION UTERUS
257
FIG. 1. Histology of Day 4.5 uterus with
embryos. A) WT (þ/þ) uterus. B) Lpar3/
uterus (/). The LE next to the embryos in
B still maintains a single layer structure, and
the stroma beneath lacks a primary decidual
zone; the presence of two adjacent embryos
in B is an early indication of embryo
crowding in Lpar3/ uterus [30]. H&E
stain, 4-lm longitudinal section of formalinfixed uterus. *Embryo; D, decidual zone.
Bar ¼ 50 lm; n ¼ 3.
RESULTS
/
Histology of Day 4.5 WT and Lpar3
Uterus
Differential Expression of Col3a1 and Col6a3 mRNA
in Day 3.5 Lpar3/ Uterus
Lpar3 expression in the WT uterus peaks at Preimplantation
Day 3.5 [30]. Microarray analysis was performed to determine
differentially expressed genes in the Day 3.5 Lpar3/ whole
uterus. Col3a1 and Col6a3 mRNA appeared more abundant in
Lpar3/ than in WT uterus, and these differential expressions
were confirmed by real-time PCR (Fig. 2). It has been
demonstrated that there is loss of COL III and COL VI (both
protein and mRNA) in the decidual zone upon implantation in
Regulation of Uterine COL III, I, VI, and IV by E2 and P4
COL III, I, VI, and IV are differentially regulated by ovarian
hormones E2 and P4 in the ovariectomized WT uterus.
Supplemental Figure S1 (all supplemental data are available
at www.biolreprod.org) shows that COL III was detected in
stroma and myometrium, but not in the epithelial compartment.
In the oil-treated control uterus, COL III fibers appeared as fine
granular staining with some connections with adjacent foci
(Supplemental Fig. S1, A and B). No obvious changes were
observed after 6 h of E2 or P4 treatment (data not shown).
However, after 24 hours of E2 (but not P4) treatment, the COL
III fibrils became thicker and longer, and appeared web-like
(Supplemental Fig. S1, C and E), which persisted after 72 h of
E2 treatment, and also showed up after 72 hours of P4
treatment (Supplemental Fig. S1, D and F). COL I staining and
regulation by E2 and P4 were similar to COL III (Supplemental
Fig. S1), except that the effects were delayed (images not
shown; see summary in Table 1). Supplemental Figure S2
shows that COL VI was detected in the intercellular spaces of
stroma and myometrium, and appeared as irregular punctuate
deposits in the oil-treated uterus (Supplemental Fig. S2, A and
B). After E2 or P4 treatment for 6 h (data not shown), a fibrillar
FIG. 2. Differential expression of Col3a1
mRNA and Col6a3 mRNA in Day 3.5 WT
and Lpar3/ uterus indicated by microarray and real-time PCR. A) Col3a1 mRNA
levels. B) Col6a3 mRNA levels. Error bars
represent standard deviation. *P , 0.05;
n ¼ 3 for microarray and n ¼ 5–6 for realtime PCR; Gapdh was used as a loading
control in the real-time PCR. Similar results
were obtained when Hprt1 was used as a
loading control in the real-time PCR (data
not shown).
Downloaded from www.biolreprod.org.
It was demonstrated previously that deletion of Lpar3 in
mice leads to delayed implantation [30]. Histological examination on Day 4.5 revealed that the WT luminal epithelium
(LE) had lost the single-layer epithelial morphology at the
implantation site, and the subepithelial stroma had undergone
morphological changes to form a primary decidual zone (Fig.
1A). In contrast, the LE opposed to blastocysts in the Lpar3/
uterus maintained a single-layer epithelium, although cells
were flattened and less dense than the adjacent LE; in addition,
no decidual changes were apparent in the subepithelial stroma
of Lpar3/ uterus (Fig. 1B). These morphological differences
suggest that the LE morphological changes and ECM
remodeling associated with early implantation may be retarded
in the Lpar3/ uterus, consistent with delayed uterine
receptivity [30].
rodents [6, 11, 34]. No other collagen genes showed
significantly differential expression in the microarray. We
therefore examined the regulation of COL III and COL VI
protein by ovarian hormones in WT uterus and their expression
in the Peri-implantation Day 0.5, 3.5, 4.5, 5.0, and 5.5 WT and
Lpar3/ uterus. We compared two other main collagen types
in the uterus—COL I and COL IV.
258
DIAO ET AL.
TABLE 1. Summary of the effects of E2 and P4 on uterine COL III, I, VI, and IV expression.a
E2
Collagen type
COL III
COL I
COL VI
COL IV
a
Effects
Thicker and
Thicker and
Thicker and
Reduced LE
longer fibrils
longer fibrils
longer fibrils (þ) and patchy fibrils (þþ)
basement membrane
P4
6h
24 h
72 h
6h
24 h
72 h
þ
þ
þ
þ
þ
þ
þ
þþ
þ
þ
þ
þ
þ/
þ
, not obvious; þ, obvious.
COL III Expression in Peri-Implantation WT
and Lpar3/ Uterus
Immunofluorescence demonstrates that, at Day 0.5, COL III
was detected throughout the uterine endometrium and
myometrium (Supplemental Fig. S4, C and D). COL III was
not detected in LE or glandular epithelial (GE) cells, but
reactivity was observed in the subepithelial ECM of both LE
and GE (Supplemental Fig. S4, C and D). A similar distribution
was observed at Day 3.5. There were no obvious differences
between WT and Lpar3/ in Day 0.5 and Day 3.5 uterus
(Supplemental Figs. S4, C and D, and S5, A and B; Fig. 3, A
and B).
Embryo implantation normally initiates around Day 4.0 in
mice. In Day 4.5 uterus, COL III showed an expression pattern
similar to Day 0.5 and Day 3.5 at low magnification (43)
(Supplemental Fig. S5, C and D), with the exception of a
seemingly lighter stain in the primary decidual zone (Supplemental Fig. S5C). At higher magnification (203), a reduced
density of COL III with some discontinuities was seen in the
stromal area in the Day 4.5 WT uterus (Fig. 3C) compared with
that of Day 3.5 (Fig. 3A). Strong COL III staining was detected
in the subepithelial ECM of LE in the WT uterus that was not
adjacent to the primary decidual zone (Fig. 3C). The COL III
stain in the subepithelial basement membrane area of LE in the
Day 4.5 Lpar3/ uterus was relatively even (Fig. 3D). The two
embryos seen next to each other in the Day 4.5 Lpar3/ uterus
is an early indication of embryo crowding in the mutant (Fig.
3D) [30].
Subsequently, there was further disappearance of COL III
from the decidual zone in Day 5.0 and Day 5.5 WT uterus, and
significant clearance of COL III was also detected in the
decidual zone of Day 5.5 Lpar3/ uterus. In addition to more
extensive COL III clearance in the Day 5.0 WT decidual zone
compared with that in the Day 4.5 WT decidual zone (Fig. 3E
and Supplemental Fig. S5E), higher magnification clearly
showed almost parallel ‘‘strings’’ of COL III in the primary
decidual zone surrounding the implanting embryo (Fig. 3E),
oriented approximately parallel to an imaginary transverse line
between mesometrium and antimesometrium (Fig. 3E and
Supplemental Fig. S5E). In Day 5.5 WT uterus, the clearance
of COL III was even more extensive, reaching almost the entire
antimesometrial endometrium, with a low level of COL III
staining still detectable in the junctional zone endometrium
(Supplemental Fig. S5G). Meanwhile, the COL III strings
present in the primary decidual zone at Day 5.0 were now
detected in both mesometrial and antimesometrial sides of the
implanting embryo, but not the decidual zone surrounding the
embryo in the Day 5.5 uterus (Fig. 3G and Supplemental Fig.
S5G). In Day 5.0 Lpar3/ uterus, uneven and strong COL III
stain in the subepithelial ECM of LE was detected (Fig. 3F and
Supplemental Fig. S5F). In Day 5.5 Lpar3/ uterus,
disappearance of COL III from the primary decidual zone
was evident (Supplemental Fig. S5H), although it was not as
extensive as that in Day 5.0 and Day 5.5 WT uterus. Similar
arrays of COL III strings to those seen in Day 5.0 WT
implantation site were observed in the Day 5.5 Lpar3/ uterus
(Fig. 3H and Supplemental Fig. S5H). Intensive COL III stain
remained in the myometrium in both the WT and Lpar3/
uterus (Supplemental Fig. S5). COL III was not detected in
embryos (Fig. 3, C–H).
COL VI Expression in Peri-Implantation WT
and Lpar3/ Uterus
In the preimplantation period (Days 0.5 and 3.5), intense
COL VI staining was detected throughout the endometrium
and myometrium (Supplemental Figs. S4, G and H, and S6, A
and B), including the vasculature. No COL VI was detected in
LE or GE, but reactivity was observed in the subepithelial
ECM of both LE and GE (Fig. 4, A and B). No obvious
difference in COL VI distribution was observed between WT
and Lpar3/ at Day 0.5 or 3.5 (Supplemental Figs. S4, G and
H, and S6, A and B).
At Day 4.5, clearance of COL VI from the implantation site
was observed in the WT uterus (Supplemental Fig. S6C), but
this was not obvious in the Lpar3/ uterus (Supplemental Fig.
S6D). Higher magnification of Day 4.5 WT implantation sites
indicated reduced COL VI staining in the primary decidual
zone; immunoreactivity remained focally strong in blood
vessel walls and adventitia, but lacked the circumferential
continuity seen on earlier days. Uneven and discontinuous
staining was also seen in the LE subepithelial ECM. No COL
VI was detected in the embryo (Fig. 4, C–H). PECAM staining
was used to visualize vascular endothelium and to confirm
COL VI localization in and adjacent to vessel walls (Fig. 4J).
These changes of COL VI distribution in decidual cells and
ECM were not observed in the Lpar3/ uterus where an
embryo was present (Fig. 4D).
At Days 5.0 and 5.5, there was further COL VI clearance
from extended areas in the WT implantation sites (Supplemental Fig. S6, E and G). Some slight evidence for clearance
could now be observed in the subepithelial stroma next to the
Downloaded from www.biolreprod.org.
web of immunoreactive COL VI became apparent and
persisted after 24 hours of E2 or P4 treatment, and 72 h of
P4 treatment (Supplemental Fig. S2, C, E, and F). However,
after 72 h of E2 treatment, COL VI staining was patchy
(Supplemental Fig. S2D), suggesting fibrillar aggregation.
Supplemental Figure S3 demonstrates the regulation of COL
IV by E2 and P4. COL IV was mainly detected in the walls of
blood vessels and in epithelial basement membranes (Supplemental Fig. S3). Upon E2 treatment, COL IV stain in the LE
basement membrane was reduced to barely detectable levels at
all three time points examined (Fig. S3, C and D, and data not
shown). However, P4 treatment did not affect COL IV
expression in basement membrane, and neither E2 nor P4
affected the distribution of COL IV in blood vessels. Table 1
summarizes the effects of E2 and P4 on the expression of
COL III, I, VI, and IV in the mouse uterus.
COLLAGEN EXPRESSION IN PERI-IMPLANTATION UTERUS
259
FIG. 3. COL III immunoreactivity in the
WT and Lpar3/ uterus from Day 3.5 to
Day 5.5. Cross sections (10 lm) were cut on
Day 3.5 and Day 5.5 uterus. Longitudinal
sections (10 lm) were cut on Day 4.5 and
Day 5.0 uterus in order to increase the
chance of obtaining sections with embryos
in the Lpar3/ uterus. A) WT, Day 3.5.
B) Lpar3/, Day 3.5. C) WT, Day 4.5. D)
Lpar3/, Day 4.5. E) WT, Day 5.0. F)
Lpar3/, Day 5.0. G) WT, Day 5.5. H)
Lpar3/, Day 5.5. Green color represents
COL III, blue represents DAPI to stain
nucleus, and the yellow star indicates the
embryo. D, decidual zone; G, glandular
epithelium; S, stroma. Bar ¼ 100 lm; n ¼
2–3.
Downloaded from www.biolreprod.org.
embryo at Day 5.0 in Lpar3/ uterus (Supplemental Fig. S6F),
and further clearance was seen at Day 5.5 (Supplemental Fig.
S6H). Higher magnification revealed disrupted COL VI
staining in the subepithelial ECM and further COL VI
breakdown in the decidualizing area in WT at Days 5.0 and
5.5 (Fig. 4, E and G). In the junctional zone endometrium of
Day 5.5 WT uterus, COL VI was still present around the
stromal cells and beneath GE basement membranes (Fig. 4I
and Supplemental Fig. S6G). Similar changes of COL VI
distribution associated with the progress of implantation were
observed in Day 5.0 and Day 5.5 Lpar3/ sites, except that
these changes were delayed compared with the same day in
WT (Fig. 4, E–H). Although COL VI reactivity disappeared
from the decidualizing cells, some staining remained in the
walls of blood vessels in implantation sites (Fig. 4, C–F, and
Supplemental Fig. S6, C–H).
COL I Expression in Peri-Implantation WT
and Lpar3/ Uterus
Like COL III, COL I is a type of fibril-forming collagen, but
microarray analysis did not show differential expression of
Col I in the Day 3.5 Lpar3/ uterus (data not shown). For
comparison, we examined COL I expression in the periimplantation WT and Lpar3/ uterus. The distribution of COL
I (Fig. 5, A and B, and Supplemental Figs. S4, A and B, and
S7, A and B) was very similar to COL III (Fig. 3, A and B, and
Supplemental Figs. S4, C and D, and S5A) at Days 0.5 and 3.5,
260
DIAO ET AL.
FIG. 4. Spatiotemporal expression of COL
VI in the WT and Lpar3/ uterus. Fresh
frozen uterine cross sections (Day 3.5 and
Day 5.5) and longitudinal sections (Day 4.5
and Day 5.0) were cut at 10 lm. A) Day 3.5
uterus to highlight LE and stroma. B) Day
3.5 uterus to highlight GE and myometrium;
there is no obvious difference in the COL VI
labeling between Day 3.5 WT and Lpar3/
uterus. C) WT, Day 4.5. D) Lpar3/, Day
4.5. E) WT, Day 5.0. F) Lpar3/, Day 5.0.
G) WT, Day 5.5. H) Lpar3/, Day 5.5.
Green color represents COL VI, blue represents DAPI, red represents PECAM, and the
yellow star indicates the embryo. B, basal
endometrium; D, decidual zone; G, glandular epithelium; Myo, myometrium; S,
stroma; V, vasculature. Bar ¼ 100 lm; n ¼
2–3.
Downloaded from www.biolreprod.org.
COLLAGEN EXPRESSION IN PERI-IMPLANTATION UTERUS
261
FIG. 5. Distribution of COL I immunoreactivity in the WT and Lpar3/ uterus from
Day 3.5 to Day 5.5. Fresh-frozen uterine
cross sections (Days 3.5 and 5.5) and
longitudinal sections (Days 4.5 and 5.0) were
cut at 10 lm. A) WT, Day 3.5. B) Lpar3/,
Day 3.5. C) WT, Day 4.5. D) Lpar3/, Day
4.5. E) WT, Day 5.0. F) Lpar3/, Day 5.0. G)
WT, Day 5.5. H) Lpar3/, Day 5.5. Blue
color indicates DAPI, green represents COL I,
and the yellow star indicates the embryo. D,
decidual zone; G, glandular epithelium; LE,
luminal epithelium; S, stroma. Bar ¼ 100
lm; n ¼ 2–3.
Downloaded from www.biolreprod.org.
and there was no obvious difference between WT and Lpar3/
uterus at either time point. As with COL III, intensive COL I
stain remained in the myometrium in both WT and Lpar3/
uterus (Supplemental Fig. S7), and COL I was not detected in
the embryos (Fig. 5, C–H).
However, compared with COL III, the clearance of COL I
from the implantation site was delayed and not as extensive
(Fig. 5, C–H, and Supplemental Fig. S7) from Day 4.5 to Day
5.5, either in WT or Lpar3/ uterus. There was discontinuous
COL I staining in Day 5.5 WT decidual zone (Fig. 5G),
whereas COL III had already disappeared in Day 5.5 WT
decidual zone (Fig. 3G). In addition, there were COL III strings
detected in the primary decidual zone at Day 5.0 (Fig. 3E), but
no obvious COL I-positive strings were observed in Day 5.0
WT implantation site (Fig. 5E), suggesting that COL I is not
part of these string structures.
COL IV Expression in the Peri-Implantation WT
and Lpar3/ Uterus
For purposes of comparison, we also examined the major
basement membrane collagen, COL IV, the mRNA level for
which was not different between Day 3.5 WT and Lpar3/
uterus in the microarray analysis (data not shown).
In the Preimplantation Days 0.5 and 3.5 uterus, COL IV was
highly expressed in the myometrium, vasculature, and the LE
and GE basement membranes, with most intensive labeling in
the vasculature; low levels were also detectable in interstitial
262
DIAO ET AL.
FIG. 6. Immunolabeling of COL IV in the
WT and Lpar3/ uterus. Fresh-frozen uterine cross sections (Days 3.5 and 5.5) and
longitudinal sections (Days 4.5 and 5.0)
were cut at 10 lm. A) Day 3.5 uterus to
highlight LE, GE, and stroma. B) Day 3.5
uterus to highlight myometrium, blood
vessel, and GE; there is no obvious difference in the COL IV labeling between Day
3.5 WT and Lpar3/ uterus. C) WT, Day
4.5. D) Lpar3/, Day 4.5. E) WT, Day 5.0.
F) Lpar3/, Day 5.0. G) WT, Day 5.5. H)
Lpar3/, Day 5.5. Green color represents
COL IV, blue represents DAPI, red represents PECAM, and the yellow star indicates
the embryo. B, basal endometrium; D,
decidual zone; G, glandular epithelium;
Myo, myometrium; S, stroma; V, vasculature. Bar ¼ 100 lm; n ¼ 2–3.
Downloaded from www.biolreprod.org.
COLLAGEN EXPRESSION IN PERI-IMPLANTATION UTERUS
Differential Expression of Serine Proteases and MMPs
in Day 3.5 Lpar3/ Uterus
Collagen turnover is regulated by various factors, including
serine proteases and MMPs [1, 35–37]. In line with the
differential expression of Col3a1 and Col6a3 in the Day 3.5
Lpar3/ uterus, microarray analysis indicated downregulation
of several genes encoding serine proteases and MMPs that have
roles in collagen degradation and ECM turnover. Differential
expression of Cma1 (mast cell chymase 1/mast cell protease 1/
mast cell protease 5/Mcp5/Mcpt5), Mcp4 (mast cell protease 4/
Mcpt4), Mcp6 (mast cell protease 6/Mcpt6), Mmp3, and Mmp7
on Day 3.5 was confirmed using real-time PCR (Fig. 7).
DISCUSSION
The regulation of COL I, III, IV, and VI by E2 and P4, as
well as the spatiotemporal expression patterns of these four
collagen types, were systemically examined in the ovariectomized and the peri-implantation mouse uterus. Each of these
collagen types has a distinct spatiotemporal regulation pattern
by E2 and P4, and a unique, dynamic pattern of expression in
the uterus in early pregnancy. All four collagen types are
expressed in the stroma and myometrium, but are undetectable
in the LE and GE during peri-implantation. COL I, III, and VI
have a discontinuous appearance in the oil-treated ovariectomized uterus. Both E2 and P4 treatments produce thicker and
longer fibrils and a web-like appearance, although, overall, the
FIG. 7. Real-time PCR confirming several genes involved in collagen
turnover indicated by microarray in Day 3.5 WT ( þ/ þ) and Lpar3/
uterus. Hprt1 is a house keeping gene used as an additional control. Error
bars represent standard deviation. *P , 0.05; n ¼ 5–6; Gapdh was used as
a loading control. Cma1, mast cell chymase 1/mast cell protease 1/mast
cell protease 5/Mcp5/Mcpt5; Mcp4, mast cell protease 4/Mcpt4; Mcp6,
mast cell protease 6/Mcpt6.
effects of E2 precede those of P4 (Table 1 and Supplemental
Figs. S1 and S2). One study indicates that the abundance of
COL I correlates with the serum E2 level, but not the P4 level,
in the naturally cycling mouse uterus [38]. The effects of E2
and P4 on COL IV are distinctly different from those on COL I,
III, and VI. E2 treatment does not affect the expression of COL
IV in the vasculature, but reduces its expression in basement
membranes, while P4 does not have an obvious effect on COL
IV expression (Supplemental Fig. S3). These results are
inconsistent with the data from natural estrous cycle, in which
the degradation of COL IV from LE basement membrane is
correlated with serum P4, but not E2 levels [38]. In
preimplantation uterus, COL I, III, and VI take on a web-like
distribution (Figs. 3–5), most likely reflecting the combined
effects of E2 and P4 on ECM (Supplemental Figs. S1 and S2).
In addition, COL IV staining in the LE basement membrane is
much weaker than in the vasculature (Fig. 6A and Supplemental Fig. S4E), whereas, in oil-treated ovariectomized
uterus, the COL IV staining in the LE basement membrane is
equally intense as that in the vasculature (Supplemental Fig.
S3, A and B), perhaps reflecting the effect of E2 on COL IV
expression in the LE basement membrane (Supplemental Fig.
S3, C and D). During peri-implantation, COL I, III, and VI
disappear from the decidual zone upon embryo implantation,
and are undetectable in the embryo (Figs. 3–5) [34]. In
contrast, COL IV accumulates in the decidual zone, and is
highly expressed in embryonic basement membranes (Fig. 6).
COL I and COL III are fibril-forming collagens. They have
the closest expression patterns among the four examined
collagen types in the WT peri-implantation uterus, and they
both have been reported to disappear from the implantation site
[5, 6]. However, the clearance of COL I from the implantation
site is not as fast and extensive as that of COL III (Figs. 3 and
5, and Supplemental Figs. S5 and S7). These results agree with
those of another study in rat uterus that shows 30% and 55%
clearance of COL I and COL III, respectively, in the
implantation site from Day 5.5 to Day 6.5; and 16% and
31% clearance of COL I and COL III, respectively, in the
implantation site from Day 6.5 to Day 7.5 [6]. Previous studies
in the mouse indicate weaker COL III staining on Day 5 than
on later days [7], with reorganization into sparsely distributed,
thick intercellular fibrils by Day 7 [39].
Downloaded from www.biolreprod.org.
areas of the endometrium. Staining was absent from LE and
GE cells. No obvious differences in COL IV expression
patterns were observed between WT and Lpar3/ at Days 0.5
or 3.5 (Fig. 6, A and B, and Supplemental Figs. S4, E and F,
and S8, A and B).
In the Day 4.5 WT uterus, reduced COL IV staining was
observed in the LE basement membrane next to the primary
decidual zone, which, in contrast, seemed to have increased
COL IV stain; faint staining was also observed in the embryo
(Fig. 6C). Although the distribution of blood vessels in the
endometrium changed upon implantation, the intensity of COL
IV reactivity in the vasculature and myometrium did not seem
to change (Fig. 6C and Supplemental Fig. S8C). These changes
were not observed in the Day 4.5 Lpar3/ uterus where an
embryo was present; in keeping with the intact epithelial layer
seen in histology (Fig. 1B), continuous LE basement
membrane staining persisted (Fig. 6D and Supplemental Fig.
S8D). Faint COL IV staining was observed in Day 4.5 embryos
(Fig. 6, C and D).
In the Day 5.0 and 5.5 WT uterus, two prominent changes
were observed: one was the appearance of intensive COL IV
staining in the primary decidual zone extending several cells
layers below the embryo; the other was increased staining in
the embryo itself (Fig. 6, E and G, and Supplemental Fig. S8, E
and G). Higher-magnification examination revealed granular
staining, both intracellular and extracellular (Fig. 6, E and G).
At Day 5.5, the embryo was intensively stained (Fig. 6G and
Supplemental Fig. S8G). These changes of COL IV staining
were not obvious in Lpar3/ uterus at Day 5.0 (Fig. 6F and
Supplemental Fig. S8F), but manifested at Day 5.5 (Fig. 6H
and Supplemental Fig. S8H), which is consistent with the effect
of delayed embryo implantation. Figure 6I shows COL IV
staining in the basal endometrium and myometrium in Day 5.5
WT uterus, with immunoreactivity mainly in the vasculature,
myometrium, and GE basement membrane. Figure 6J confirms
that COL IV and PECAM were coexpressed in the blood
vessels, but not other areas.
263
264
DIAO ET AL.
myometrium may be involved in the pathogenesis of severe
pre-eclampsia [43]. Reduced COL IV expression in human
decidual tissues may be associated with spontaneous abortion
[44]. Reduced COL IV expression in the endometrium of
women postluteinizing hormone surge was associated with
unexplained infertility [45]. Decreased levels of MMP9 and
tissue inhibitor of MMP (TIMP) 1 were detected in uterine
fluid during the expected implantation window in women with
infertility and unexplained recurrent miscarriage [46]. Decreased uterine receptivity is a main reason for the low
pregnancy rate and higher miscarriage rate in patients with
polycystic ovary syndrome (PCOS). It has been demonstrated
that Mmp26 is downregulated in the uterus of PCOS patients
during the expected implantation window [47]. Compared to
normal midsecretory endometrium (expected implantation
window), higher mRNA levels of Col I, Mmp2, and cathepsin
H, as well as lower mRNA levels of Timp3 were detected in
midsecretory endometrium of patients with unexplained
infertility and/or recurrent miscarriages [48].
This study demonstrates that deletion of Lpar3 leads to
altered spatiotemporal expression of COL I, III, IV, and VI in
the peri-implantation uterus. These changes are in line with the
delayed uterine receptivity in the Lpar3/ uterus. However,
the functional significance of these collagen types in the
Lpar3/ mice is currently unknown. We speculate that altered
collagen expression could contribute to delayed implantation
and subfertility in Lpar3/ mice.
ACKNOWLEDGMENTS
The authors thank Dr. James N. Moore and Dr. Zhen Fu at the College
of Veterinary Medicine, University of Georgia for the access to the ABI
7900 Real-Time PCR machine and the imaging system, respectively, the
University of Georgia Center for Advanced Ultrastructural Research for
access to the fluorescent microscope, and the Genomics and Proteomics
Core Facility Medical College of Georgia for the microarray analysis.
REFERENCES
1. Aplin JD. Endometrial extracellular matrix. In: Aplin JD, Fazleabas AT,
Glasser SR, and Giudice LC (eds.), The Endometrium: Molecular,
Cellular, and Clinical Perspectives, 2nd ed. London, United Kingdom:
Informa Healthcare; 2008:364–378.
2. Heino J. The collagen family members as cell adhesion proteins.
Bioessays 2007; 29:1001–1010.
3. Morrione TG, Seifter S. Alteration in the collagen content of the human
uterus during pregnancy and post partum involution. J Exp Med 1962;
115:357–365.
4. Hurst PR, Gibbs RD, Clark DE, Myers DB. Temporal changes to uterine
collagen types I, III and V in relation to early pregnancy in the rat. Reprod
Fertil Dev 1994; 6:669–677.
5. Clark DE, Hurst PR, McLennan IS, Myers DB. Immunolocalization of
collagen type I and laminin in the uterus on Days 5 to 8 of embryo
implantation in the rat. Anat Rec 1993; 237:8–20.
6. Hurst PR, Palmay RD, Myers DB. Localization and synthesis of collagen
types III and V during remodelling and decidualization in rat uterus.
Reprod Fertil Dev 1997; 9:403–409.
7. Spiess K, Teodoro WR, Zorn TM. Distribution of collagen types I, III, and
V in pregnant mouse endometrium. Connect Tissue Res 2007; 48:99–108.
8. Armant DR, Kameda S. Mouse trophoblast cell invasion of extracellular
matrix purified from endometrial tissue: a model for peri-implantation
development. J Exp Zool 1994; 269:146–156.
9. Blankenship TN, Given RL. Loss of laminin and type IV collagen in
uterine luminal epithelial basement membranes during blastocyst implantation in the mouse. Anat Rec 1995; 243:27–36.
10. Farrar JD, Carson DD. Differential temporal and spatial expression of
mRNA encoding extracellular matrix components in decidua during the
peri-implantation period. Biol Reprod 1992; 46:1095–1108.
11. Mulholland J, Aplin JD, Ayad S, Hong L, Glasser SR. Loss of collagen
type VI from rat endometrial stroma during decidualization. Biol Reprod
1992; 46:1136–1143.
Downloaded from www.biolreprod.org.
The expression pattern of COL IV differs from that of the
other three collagen types. It has been previously reported that
COL IV disappears from the LE basement membrane and
appears in the embryo during implantation [9], which is
consistent with our results (Fig. 6 and Supplemental Fig. S8).
The accumulation of COL IV protein in the decidual zone
(Fig. 6 and Supplemental Fig. S8) is also consistent with the
reported Col4a1 mRNA and COL IV protein expression in
the same area [9, 10]. COL IV consistently has intensive
expression in the blood vessels in the peri-implantation mouse
uterus (Fig. 6 and Supplemental Fig. S8). COL VI is also
detected in the blood vessels, but its relative expression level
is not as intensive as that of COL IV when compared with the
expression levels in other uterine compartments. Interestingly,
COL VI degradation in decidua is spatially restricted, not
extending to the vessel walls (Fig. 4 and Supplemental Fig.
S6). A similar phenomenon occurs in both rat and human
decidua [11, 40]. This probably reflects the role of COL VI,
which seems to envelop vessels, perhaps as a way of
integrating them into the surrounding three-dimensional tissue
architecture.
These spatiotemporal changes in COL I, III, IV, and VI in
the Lpar3/ peri-implantation mouse uterus are all delayed
compared with that in the WT peri-implantation uterus (Figs.
3–6, and Supplemental Figs. S5–S8). These changes were
different from the effects of E2 and P4 on uterine collagen
expression in ovariectomized WT uterus, indicating that the
altered collagen expression in Lpar3/ uterus is unlikely to
be a result of alterations in ovarian hormones. The statement
is supported by our observation that there is no significant
difference in serum E2 and P4 levels between WT and
Lpar3/ mice on Day 3.5 and Day 4.5 (data not shown).
One explanation for retention of COL III and COL VI in the
Lpar3/ implantation sites could be higher mRNA expression levels (Fig. 2), leading to elevated rates of protein
synthesis and, possibly, more abundant ECM collagen,
although this was not apparent in the immunostaining at
Day 3.5. In addition, decreased uterine expression of genes
involved in ECM turnover, including mast cell proteases and
MMPs (Fig. 7), may very well contribute importantly to the
delayed clearance of COL I, III, and VI in Lpar3/ uterus
[20, 37]. COL VI is resistant to some of the major MMPs;
however, it can be degraded by mast cell chymases and some
other serine proteinase matrix-degrading enzymes [21]. Mast
cell chymase can also regulate the activities of pro-MMP-2
and -9; as well as inhibit the expression of Col I and Col III
mRNA [23, 41]. It seems that COL IV is synthesized in the
decidual cells [9, 10], so the delayed accumulation of COL
IV may reflect delayed implantation in Lpar3/ uterus (Fig.
6 and Supplemental Fig. S8), with delayed onset of de novo
gene expression and production of COL IV in the decidual
zone.
How is LPA3 involved in regulating collagen metabolism?
Lpar3 is mainly expressed in the LE [30], where none of the
collagen types that we have examined are detected (Fig. 3–6)
[34]. Thus, LE may not be a main source for collagen synthesis
in the period under study. The LE-specific localization of
Lpar3 suggests that LPA3 may have a paracrine effect on
collagen regulation [42]. Since LE is the first layer of cells with
which an implanting embryo communicates, if LPA3 mediates
the communication between an implanting embryo and the
stromal cells, its absence could lead to a delay in the molecular
program of primary decidual differentiation.
Dysregulation of uterine ECM turnover has been associated
with uterine dysfunction, including defective uterine receptivity. Overproduction of human mast cell chymase in the
COLLAGEN EXPRESSION IN PERI-IMPLANTATION UTERUS
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
Amano T, Kennedy G, Arai H, Aoki J, Chun J. LPA3-mediated
lysophosphatidic acid signalling in embryo implantation and spacing.
Nature 2005; 435:104–108.
Hama K, Aoki J, Inoue A, Endo T, Amano T, Motoki R, Kanai M, Ye X,
Chun J, Matsuki N, Suzuki H, Shibasaki M, Arai H. Embryo spacing and
implantation timing are differentially regulated by LPA3-mediated
lysophosphatidic acid signaling in mice. Biol Reprod 2007; 77:954–959.
Pelch KE, Schroder AL, Kimball PA, Sharpe-Timms KL, Davis JW,
Nagel SC. Aberrant gene expression profile in a mouse model of
endometriosis mirrors that observed in women. Fertil Steril 2010; 93:
1615–1627.
Ye X, Skinner MK, Kennedy G, Chun J. Age-dependent loss of sperm
production in mice via impaired lysophosphatidic acid signaling. Biol
Reprod 2008; 79:328–336.
Dziadek M, Darling P, Zhang RZ, Pan TC, Tillet E, Timpl R, Chu ML.
Expression of collagen alpha 1(VI), alpha 2(VI), and alpha 3(VI) chains in
the pregnant mouse uterus. Biol Reprod 1995; 52:885–894.
Song F, Wisithphrom K, Zhou J, Windsor LJ. Matrix metalloproteinase
dependent and independent collagen degradation. Front Biosci 2006; 11:
3100–3120.
Trackman PC. Diverse biological functions of extracellular collagen
processing enzymes. J Cell Biochem 2005; 96:927–937.
Lauer-Fields JL, Juska D, Fields GB. Matrix metalloproteinases and
collagen catabolism. Biopolymers 2002; 66:19–32.
Wood GA, Fata JE, Watson KL, Khokha R. Circulating hormones and
estrous stage predict cellular and stromal remodeling in murine uterus.
Reproduction 2007; 133:1035–1044.
Spiess K, Zorn TM. Collagen types I, III, and V constitute the thick
collagen fibrils of the mouse decidua. Microsc Res Tech 2007; 70:18–25.
Aplin JD, Charlton AK, Ayad S. An immunohistochemical study of
human endometrial extracellular matrix during the menstrual cycle and
first trimester of pregnancy. Cell Tissue Res 1988; 253:231–240.
Wang Y, Shiota N, Leskinen MJ, Lindstedt KA, Kovanen PT. Mast cell
chymase inhibits smooth muscle cell growth and collagen expression in
vitro: transforming growth factor-beta1-dependent and -independent
effects. Arterioscler Thromb Vasc Biol 2001; 21:1928–1933.
Xie Y, Gibbs TC, Meier KE. Lysophosphatidic acid as an autocrine and
paracrine mediator. Biochim Biophys Acta 2002; 1582:270–281.
Mitani R, Maeda K, Fukui R, Endo S, Saijo Y, Shinohara K, Kamada M,
Irahara M, Yamano S, Nakaya Y, Aono T. Production of human mast cell
chymase in human myometrium and placenta in cases of normal
pregnancy and preeclampsia. Eur J Obstet Gynecol Reprod Biol 2002;
101:155–160.
Iwahashi M, Muragaki Y, Ooshima A, Nakano R. Decreased type IV
collagen expression by human decidual tissues in spontaneous abortion. J
Clin Endocrinol Metab 1996; 81:2925–2929.
Bilalis DA, Klentzeris LD, Fleming S. Immunohistochemical localization
of extracellular matrix proteins in luteal phase endometrium of fertile and
infertile patients. Hum Reprod 1996; 11:2713–2718.
Skrzypczak J, Wirstlein P, Mikolajczyk M. Could the defects in the
endometrial extracellular matrix during the implantation be a cause for
impaired fertility? Am J Reprod Immunol 2007; 57:40–48.
Qiao J, Wang L, Li R, Zhang X. Microarray evaluation of endometrial
receptivity in Chinese women with polycystic ovary syndrome. Reprod
Biomed Online 2008; 17:425–435.
Jokimaa V, Oksjoki S, Kujari H, Vuorio E, Anttila L. Altered expression
of genes involved in the production and degradation of endometrial
extracellular matrix in patients with unexplained infertility and recurrent
miscarriages. Mol Hum Reprod 2002; 8:1111–1116.
Downloaded from www.biolreprod.org.
12. Rahima A, Soderwall AL. Uterine collagen content in young and
senescent pregnant golden hamsters. J Reprod Fertil 1977; 49:161–162.
13. Ryan JN, Woessner JF Jr. Oestradiol inhibits collagen breakdown in the
involuting rat uterus. Biochem J 1972; 127:705–713.
14. Ryan JN, Woessner JF Jr. Oestradiol inhibition of collagenase role in
uterine involution. Nature 1974; 248:526–528.
15. Koob TJ, Jeffrey JJ. Hormonal regulation of collagen degradation in the
uterus: inhibition of collagenase expression by progesterone and cyclic
AMP. Biochim Biophys Acta 1974; 354:61–70.
16. Pan Y, Liu Z, Zhu C. Effect of mifepristone on the expression of chorionic
gonadotropin beta subunit and collagen type IV in female rhesus monkey
decidua and villus at early gestation [in Chinese]. Zhonghua Nan Ke Xue
2004; 10:358–361, 365.
17. Rodriguez HA, Kass L, Varayoud J, Ramos JG, Ortega HH, Durando M,
Munoz-De-Toro M, Luque EH. Collagen remodelling in the guinea-pig
uterine cervix at term is associated with a decrease in progesterone
receptor expression. Mol Hum Reprod 2003; 9:807–813.
18. Rodriguez HA, Ramos JG, Ortega HH, Munoz-de-Toro M, Luque EH.
Regional changes in the spatio-temporal pattern of progesterone receptor
(PR) expression in the guinea-pig genital tract as parturition approaches. J
Steroid Biochem Mol Biol 2008; 111:247–254.
19. Alexander CM, Hansell EJ, Behrendtsen O, Flannery ML, Kishnani NS,
Hawkes SP, Werb Z. Expression and function of matrix metalloproteinases and their inhibitors at the maternal-embryonic boundary during
mouse embryo implantation. Development 1996; 122:1723–1736.
20. Curry TE Jr, Osteen KG. The matrix metalloproteinase system: changes,
regulation, and impact throughout the ovarian and uterine reproductive
cycle. Endocr Rev 2003; 24:428–465.
21. Kielty CM, Lees M, Shuttleworth CA, Woolley D. Catabolism of intact
type VI collagen microfibrils: susceptibility to degradation by serine
proteinases. Biochem Biophys Res Commun 1993; 191:1230–1236.
22. Kofford MW, Schwartz LB, Schechter NM, Yager DR, Diegelmann RF,
Graham MF. Cleavage of type I procollagen by human mast cell chymase
initiates collagen fibril formation and generates a unique carboxyl-terminal
propeptide. J Biol Chem 1997; 272:7127–7131.
23. Tchougounova E, Lundequist A, Fajardo I, Winberg JO, Abrink M, Pejler
G. A key role for mast cell chymase in the activation of pro-matrix
metalloprotease-9 and pro-matrix metalloprotease-2. J Biol Chem 2005;
280:9291–9296.
24. Abe M, Ho CH, Kamm KE, Grinnell F. Different molecular motors
mediate platelet-derived growth factor and lysophosphatidic acid-stimulated floating collagen matrix contraction. J Biol Chem 2003; 278:47707–
47712.
25. Watterson KR, Lanning DA, Diegelmann RF, Spiegel S. Regulation of
fibroblast functions by lysophospholipid mediators: potential roles in
wound healing. Wound Repair Regen 2007; 15:607–616.
26. Sato M, Shegogue D, Hatamochi A, Yamazaki S, Trojanowska M.
Lysophosphatidic acid inhibits TGF-beta-mediated stimulation of type I
collagen mRNA stability via an ERK-dependent pathway in dermal
fibroblasts. Matrix Biol 2004; 23:353–361.
27. Fishman DA, Liu Y, Ellerbroek SM, Stack MS. Lysophosphatidic acid
promotes matrix metalloproteinase (MMP) activation and MMP-dependent invasion in ovarian cancer cells. Cancer Res 2001; 61:3194–3199.
28. Mutoh T, Chun J. Lysophospholipid activation of G protein-coupled
receptors. Subcell Biochem 2008; 49:269–297.
29. Choi JW, Herr DR, Noguchi K, Yung YC, Lee CW, Mutoh T, Lin ME,
Teo ST, Park KE, Mosley AN, Chun J. LPA receptors: subtypes and
biological actions. Annu Rev Pharmacol Toxicol 2010; 50:157–186.
30. Ye X, Hama K, Contos JJ, Anliker B, Inoue A, Skinner MK, Suzuki H,
265